Overview of Bone Grafts
Rapid and effective repair of bone defects caused by injury, disease, wounds, or surgery is a goal of orthopaedic surgery. Toward this end, a number of compositions and materials have been used or proposed for use in the repair of bone defects. The biological, physical, and mechanical properties of the compositions and materials are among the major factors influencing their suitability and performance in various orthopaedic applications.
Bone includes various types of cells and an abundant mineralized extracellular matrix. Bone resorption and bone formation are the processes involved in normal body morphogenesis and calcium homeostasis in the body. In addition to its physiological role, bone resorption plays roles in pathological disorders such as osteoporosis, metabolic bone diseases, bone fracture, and malignant hypercalcemia.
Autologous cancellous bone (“ACB”), also known as autograft or autogenous bone, has been considered the gold standard for bone grafts. ACB includes osteogenic cells, which have the potential to assist in bone healing, is nonimmunogenic, and has structural and functional characteristics that should be appropriate for a healthy recipient. Some people do not have adequate amounts of ACB for harvesting. These people include, for example, older people and people who have had previous surgeries. Some individuals lack ACB of appropriate dimensions and quality for transplantation, and donor site pain and morbidity associated with the harvesting of ACB can pose serious problems for patients and their physicians.
Much effort has been invested in the identification and development of alternative bone graft materials. Urist published seminal articles on the theory of bone induction and a method for decalcifying bone, i.e., making demineralized bone matrix (DBM). Urist M. R., Bone Formation by Autoinduction, Science 1965; 150 (698):893-9; Urist M. R. et al., The Bone Induction Principle, Clin. Orthop. Rel. Res. 53:243-283, 1967. DBM is an osteoinductive material in that it induces bone growth when implanted in an ectopic site of a rodent, owing to the osteoinductive factors contained within the DBM. Honsawek et al. (2000). It is now known that there are numerous osteoinductive factors, e.g., BMP 1-18 (Bone Morphogenetic Protein), which are part of the transforming growth factor-beta (TGF-beta) superfamily. BMP-2 has been widely studied. There are also other proteins present in DBM that are not osteoinductive alone but still contribute to bone growth, including fibroblast growth factor-2 (FGF-2), insulin-like growth factor-I and -II (IGF-I and IGF-II), platelet derived growth factor (PDGF), and transforming growth factor-beta 1 (TGF-beta.1) (Hauschka, et al. 1986; Canalis, et al, 1988; Mohan et al. 1996).
Bone grafting applications are differentiated by the requirements of the skeletal site. Certain applications require a “structural graft” and other applications require an “osteogenic graft.” These requirements are not mutually exclusive and some applications may benefit from a structural, osteogenic graft. Grafts may also have other beneficial biological properties, such as, for example, serving as delivery vehicles for bioactive substances. Bioactive substances include physiologically or pharmacologically active substances that act locally or systemically in the host.
A structural graft is a graft in which one role of the graft is to provide mechanical or structural support at the surgical site. Such grafts may contain a substantial portion of mineralized bone tissue to provide the strength needed to be load-bearing. Examples of applications requiring a structural graft include intercalary grafts, spinal fusion, joint plateaus, joint fusions, large bone reconstructions, etc. The biomechanical properties of osteoimplants upon implantation are determined by many factors, including the specific source of the bone used to make the osteoimplant; various physical characteristics of donor tissue; and the method chosen to prepare, preserve, and store the bone prior to implantation, as well as the type of loading to which the graft is subjected.
Mineralized bone may be used in osteoimplants in part because of its inherent strength, i.e., its load-bearing ability at the recipient site. Structural osteoimplants are conventionally made by processing, and then machining or otherwise shaping cortical bones collected for transplant purposes. Osteoimplants may comprise monolithic bone of an aggregate of particles. Further, osteoimplants may be substantially solid, flowable, or moldable. Cortical bone can be configured into a wide variety of configurations depending on the particular application for the structural osteoimplant. Structural osteoimplants are often provided with intricate geometries, e.g., series of steps; concave or convex surfaces; tapered surfaces; flat surfaces; surfaces for engaging corresponding surfaces of adjacent bone, tools, or implants, hex shaped recesses, threaded holes; serrations, etc.
An osteogenic graft is a graft in which one role of the graft is to enhance or accelerate the growth of new bone tissue at the site. Such grafts may contain demineralized bone tissue to improve the osteoinductivity needed for growth of new bone tissue. Examples of applications requiring “osteogenic graft” include deficit filling, spinal fusions, joint fusions, etc.
Bone healing, or remodeling, generally comprises a multi-step process including resorption of existing bone by osteoclasts, formation of new blood vessels, and the subsequent growth of new bone by osteoblasts. Bone resorption and bone formation are thus linked. Bone resorption is generally determined by the rate of osteoclast recruitment and the intensity and duration of osteoclast activity. Starting with osteoclast recruitment, osteoclasts are derived from hemopoietic precursors, for example CFU-M branching off the monocyte macrophage lineage. Agents that stimulate bone resorption in vivo and increase osteoclast formation from bone marrow cultures in vitro and have been implicated in the pathogenesis of osteoporosis include parathyroid hormone and, under certain conditions, IL-6. After bone resorption, the bone remodeling cycle continues into bone formation, using osteoblastic cells.
Osteoclasts function to resorb mineralized bone, dentine, and calcified cartilage. An overview of osteoclast origins and function can be found in “The Osteoclast” (Bone, Vol. 2, B. K. Hall, ed., CRC Press, 1991, 272 pages), incorporated herein by reference. Both mononuclear and multinuclear osteoclasts can resorb bone. Osteoclasts are blood-borne cells originating from hemopoietic mononuclear stem cells or hemopoietic progenitors. The progenitors express some osteoclast-specific genes and proteins, fuse with each other, and differentiate into functionally mature osteoclasts. The differentiation and function of osteoclasts generally are controlled by various osteotropic hormones and local factors. These factors act on osteoclasts and their precursors directly or indirectly via other bone cells. Osteoclasts resorb both the mineral and the organic phases of bone. They generally contain between approximately 1 and approximately 50 nuclei, and range from approximately 20 to over approximately 200 micrometers in diameter. In trabecular bone, they occupy shallow excavations on the surface, and in Haversion bone, they occupy the leading edge of cutting cones. Light microscopic features include irregular cell shape, foamy, acidophilic cytoplasm, a striated perimeter zone of attachment to the bone, and positive staining for tartrate-resistant acid phosphatase. Electron microscopic features are numerous mitochondria, rough endoplasmic reticulum, multiple Golgi complexes, pairs of centrioles in a centrosome, vacuoles, and numerous granules. A ruffled border is located at the interface between resorbing bone surface and the cell surface. Osteoclasts secrete collagenase and acid phosphatase. Carbonic anhydrase is utilized for formation of H+ ions secreted at the ruffled border. The life span of osteoclast work at a trabecular resorption site is about four weeks on average.
Osteoblasts are bone-forming cells. An overview of osteoblast origins and function can be found in “The Osteoblast and Osteocyte” (Bone, Vol. 1, B. K. Hall, ed., CRC Press, 1991, 494 pages), incorporated herein by reference. They produce the organic collagen matrix (and noncollagenous proteins) that undergoes mineralization to form both lamellar and woven bone. Osteoblasts generally originate from marrow stromal cell lineage and appear at bone remodeling sites where osteoclasts previously resorbed bone. Prominent features of osteoblasts are an eccentric nucleus, Golgi apparatus, cell processes, gap junctions, endoplasmic reticulum, and collagen secretory granules.
In healthy bone, bone remodeling, including resorption and formation, is a continuous process. That is, bone resorption, followed by bone formation, occurs continually and in a balanced fashion. More specifically, bone remodeling comprises erosion of bone by osteoclasts, followed by resorption of bone, followed by new bone formation. Overall, each of these processes are part of a surface-based process.
A regulatory system that keeps bone resorption and formation in balance is the RANKL/OPG system (the receptor activator for nuclear factor κ B ligand and osteoprotegerin (OPG) regulatory system). RANKL, a 316-amino acid transmembrane protein, is highly expressed by osteoblast/stromal cells in cancellous (or trabecular) bone. RANKL binds as a homotrimer to RANK (the receptor activator for nuclear factor κ B), a 616-amino acid transmembrane receptor (also a trimer) on the surface of monocyte/macrophage lineage cells, including osteoclasts, and their precursors (pre-osteoclasts). RANKL stimulates osteoclast activity by generating multiple intracellular signals that regulate cell differentiation, function, and survival, such as via ADAM proteins (a disintegrin and metalloprotease). Thus, osteoblasts (bone forming cells) stimulate and contribute to the formation of osteoclasts (cells that break down bone).
Osteoblasts further have a role in regulating formation of osteoclasts. While osteoblasts stimulate osteoclasts by producing and expressing RANKL, osteoblasts also regulate osteoclast formation by secreting OPG, a 380-amino acid-soluble receptor that binds to RANKL and blocks it. Generally, OPG inhibits formation of osteoclasts. In addition to expression by osteoblasts, OPG is expressed by stromal, cardiovascular, and other cells.
Fully differentiated osteoclasts on a bone surface can begin to resorb bone in response to a variety of stimuli, such as for example hormones, cytokines, or adhesion molecules present in the bone matrix or on membranes of other bone cells. The bone-resorbing activity of osteoclasts can be enhanced by some factors produced by osteoblastic UMR cells in response to 1,25(OH)2D3 (Vitamin D), PTH (Parathyroid Hormone), and PTHrP (Parathyroid hormone-related Peptide). Because of the impurity and insufficient number of osteoclasts, investigation of osteoclast activity is generally limited to single-cell studies involving procedures such as electrophysiology, immunocytochemistry, histochemistry, and single-cell molecular techniques.
In the event of trauma to bone, such as from injury or surgery, a wound is created. This disrupts the normal balance of bone turnover to favor bone growth over resorption. The body's initial response to a wound is inflammation, which leads to a temporary increase in the rate of bone resorption through stimulated osteoclast activity as well as generalized macrophage activity (macrophage activity generally being confined to bone particles).
One mechanism for stimulating resorption is through secretion of macrophage colony stimulating factors (MCSF). MCSF can be secreted by various cell types, including adipocytes, vascular endothelial cells, and smooth muscle cells. In combination with RANKL, MCSF can stimulate production of new osteoclasts from osteoclast precursors circulating in the blood. Thus, bone resorption is stimulated by the presence of the additional MCSF induced osteoclasts.
Various other regulating proteins may affect the resorption/formation balance by stimulating expression of RANKL, secretion of MCSF, inhibiting the secretion of OPG (by PTH (parathyroid hormone), for example), and/or carrying out the developmental cascade of cellular events initiated by RANKL and MCSF. Examples of such regulating proteins include, without limitation, ADAM-12 (a disintegrin and metalloprotease-12); PTH; PTHrP; VEGF (vascular endothelial growth factor); Hydrocortisone; 1, 25 dihydroxyvitamin D3; PGE2 (prostaglandin E2); TNFalpha (tumor necrosis factor-alpha); IL-1beta (Interleukin-3 beta), IL-3, IL-6; and bFGF (basic fibroblast growth factor).
Certain types of bone grafts are known to remodel at a slow rate. For example, structural grafts are known to remodel over a period of several years. Increasing the rate of the bone healing process is thus especially beneficial for such types of bone grafts. Conventional approaches to increasing the rate of bone healing, such as those employing demineralized bone and/or growth factors, concentrate primarily on increasing osteoblast activity, i.e., increasing the rate of bone formation. Such approaches do not directly take advantage of the impact of resorption and remodeling in the bone healing process. Accordingly, to stimulate the bone resorption rate, and thus the overall rate of bone healing, osteoclast activity may be stimulated by supplying MCSF, RANKL, and/or various other regulating proteins to a wound site.